Yl disulfonate stable free radical structures prepared from serum albumin and nitros

ABSTRACT

SUBJECT DISCLOSURE RELATES TO A STABLE FREE-RADICAL PRODUCT, ITS USE TO PRODUCE BRAIN EXCITATION IN MAMMALS WHEN INTRAVENOUSLY ADMINISTERED, AND METHOD OF PREPARING THE FREE-RADICAL PRODUCT. THE FREE-RADICAL PRODUCT IS PREPARED FROM PURIFIED PLASMA PROTEINS, PITUITARY PEPTIDES AND OTHER STRUCTURES CONTAINING TYROSINE OR TRYPTOPHAN BY OXIDATION WITH A FREE-RADICAL NITROSYL DISULFONATE PRODUCT IN ALKALINE SOLUTION UNDER CONTROLLED CONDITIONS. INTRAVENOUS ADMINISTRATION OF THE FREE-RADICAL PRODUCT INTO MAMMALS CAUSES A SUDDEN ELECTROENCEPHALOGRAPHIC AROUSAL ACCOMPANIED BY BEHAVIORAL CHANGES INDICATIVE OF BRAIN EXCITATION. ILLUMINATION OF THE FREE-RADICAL PRODUCT BY VISIBLE LIGHT PRIOR TO ADMINISTRATION SUBSTANTIALLY ENHANCES THE AROUSAL AND BRAIN EXTRACTION EFFECTS.

United States Patent US. Cl. 260-121 1 Claim ABSTRACT OF THE DISCLOSURESubject disclosure relates to a stable free-radical product, its use toproduce brain excitation in mammals when intravenously administered, anda method of preparing the free-radical product. The free-radical productis prepared from purified plasma proteins, pituitary peptides and otherstructures containing tyrosine or tryptophan by oxidation with afree-radical nitrosyl disulfonate product in alkaline solution undercontrolled conditions. Intravenous administration of the free-radicalproduct into mammals causes a sudden electroencephalographic arousalaccompanied by behavioral changes indicative of brain excitation.Illumination of the free-radical product by visible light prior toadministration substantially enhances the arousal and brain excitationeffects.

This is a continuation-in-part application of my copending applicationSer. No. 74,487, filed Sept. 22, 1970, now abandoned.

The invention described herein may be manufactured and used by or forthe Government of the United States of America for governmental purposeswithout the payment of any royalties thereon or therefor.

In the various chemical reactions of living systems, free energy isoften exchanged by the transformation of electronic energy present inchemical bonds. In the photobiological and radiobiological arts, theelectron excited state or the free-radical form of substances plays animportant role in transformation reactions. Similarly, it is believedthat the free-radical or electron excited state of the substance playsan important role in transformation reactions of mammalian biologicalsystems. Although various substances have been developed in the past toproduce brain arousal and brain excitatory effects in mammals, fewrelatively stable free-radical substances are known which can beinjected into the animal system for that purpose.

It is therefore a principal object of the invention provide a novel andimproved substance which is relatively stable in its free-radical stateand which is particularly useful in inducing arousal and brainexcitatory effects in mammals.

It is a further object of the invention to convert molecules of anaturally occurring substance to a stable freeradical state such thatupon injection into a mammalian system they modify the brain function inan energy transfer reaction.

Further objects and advantages of the invention will be apparenthereinafter.

In accordance with the invention, unique relatively stable free-radicalsubstances are injected into intact mammals to produce aneurophysiologically excited state which is apparently caused by thetransfer of the energy of electron excited molecules to structures thatinduce brain excitatory effects. The stable free-radical substances areprepared from crystalline albumin, purified fractions of serum proteins,hormonal peptides and other structures which contain tyrosine ortryptophan by reacting Patented Sept. 11, 1973 the same with nitrosyldisulfonate in a slightly alkaline solution at a reduced temperaturesuch as 5 C. using 2 to 4 moles of nitrosyl disulfonate per mole of thetyrosine or tryptophan containing substance. The presence of either thetyrosine or tryptophan amino acid in the compound was found to be aprerequisite for the stable free-radical formation. The free-radical wasfound to be stable for weeks in solution at 5 C. and has been maintainedin its free radical state for nearly a year at 20 C. Illumination of thefree-radical compounds by visible light for 1-5 minutes immediatelyprior to their administration to the mammal substantially enhanced theireffect. Various stable free-radical substances containing tyrosine ortryptophan were prepared under substantially similar conditions withvarying final steps for separation of the free-radical from otherreaction products and excess reactants to suit the properties of theprotein or peptide. More specifically, stable free radical moleculeswere obtained from such substances as or and ,B globulins, bovine growthhormone, thyrotrophin, glucagon, follicle stimulating hormone,luteinizing hormone, adrenotrophin, insulin, thyroxin, melatonin andS-OH tryptophan.

Specific and preferred embodiments of the improved free-radicalsubstance of the invention are illustrated by the following examples:

EXAMPLE 1 Three grams of 3X recrystallized bovine serum albumin weredissolved in ml. of saline-phosphate buffer (.15 M NaCl, .01 M P0 pH8.1). Solutions of nitrosyl disulfonate (NDS) were prepared from Fremysalt having the formula [ON(SO K by dissolving .8 gram of the salt in 45ml. of .1 M N HPQ, and diluting to 100 ml. with water. The final pH was7.5. This formed a fairly stable solution of NDS free-radical.Concentrations were determined from optical density measurements at 545millimicrons using 20.8 as the molar extinction coefficient. The albuminsolution was mixed with an equal volume of the .025 M NDS solution.After standing overnight at 5 (3., the purple color of the NDSdisappeared and the protein became colored a deep burgundy red. Theprotein was separated from reaction products and excess NDS byprecipitation with 3.6 M ammonium sulfate and subsequent dialysis toremove the salt and by chromatography. The protein free-radical wasstable for weeks in solution at 5 C. Samples were maintained in theirfreeradieal state for nearly a year when frozen at 20 C.

EXAMPLE 2 2 grams of rabbit alpha globulin Cohn Fraction IV-l weredissolved in 90 ml. of saline-Na HPO buffer. 52.53 ml. of .03173 M NDSwere then mixed with the globulin solution and the mixture was dilutedto a total volume of 166 ml. The resulting free-radical product, whichcontained 1.204% protein, was separated from excess reactants and otherreaction products by methods similar to those described in Example 1.

EXAMPLE 3 2 grams of rabbit beta globulin FR 111 were dissolved in 90ml. of saline-Na HPQ, buffer. 52.53 ml. of .03173 M NDS were then mixedwith the globulin solution and the mixture was diluted to a total volumeof 153 ml. The resulting free-radical product, which contained 1.307%protein, was separated from excess reactants and other reaction productsby methods similar to those described in Example 1.

EXAMPLE 4 mg. of bovine growth hormone having a pH of 8.2 and 1% proteinwas diluted to 30 ml. with .1 M Na HPO 20ml. of .025 M NDS were thenmixed with the hormone solution to form the desired free-radicalproduct. The

free-radical product was separated from excess reactants and otherreaction products by centrifuge and dialysis methods similar to thosedescribed in Example 1.

EXAM PLE 5 50 mg. of glucagon was dissolved with a little NaOH in a P0,;buffer. The pH of the solution was approximately but was immediatelytaken down to 8 with HCl. 2 ml. of .025 M NDS were thin mixed with theglucagon solution to form the desired free-radical product. Thefree-radical product was separated out and concentrated by passing thesame through a UM3 Diaflow Ultrafilter.

EXAMPLE 6 202.1 mg. of adrenotrophin was placed in solution to a totalvolume of 7.4 ml. The solution had a pH of 6.7. 11 ml. of .025 M NDSwere then mixed with the adrenotrophin solution in a cold room for 24hours to form the desired free-radical product. The free-radical productwas separated from excess reactants and other reaction products bymethods similar to those described in Example 1.

EXAMPLE 7 '100 mg. of old crystalline zinc insulin was dissolved in 2ml. of .025 M NDS to form the desired free-radical product. Thefree-radical product was separated from excess reactants and otherreaction products and concentrated on an ultrafilter.

EXAMPLE 8 10 ml. of thyroxine (.001 M in .005 M NaOH) was combined with1 ml. of .025 M NDS and left at room temperature for 2 hours. Thesolution was then taken to a pH of 3 with .5 M H 80 The resultingfree-radical product was precipitated in alcohol and separated fromexcess reactants and other reaction products by methods similar to thosedescribed in Example 1.

EXAMPLE 9 One volume of .047 M 5 hydroxytryptophan was mixed with twovolumes of .025 M NDS to form the desired free-radical product. Thefree-radical product was separated from excess reactants and otherreaction products by methods similar to those described in Example 1.Inasmuch as this free-radical product was relatively unstable, the samewas used in its various mammalian experiments described hereinafterimmediately after its preparation.

Various tests and experiments were performed using the free-radicalsubstances obtained in the above described examples. Visible andultraviolet absorption spectra of the free-radical substances wereobtained with a Cary spectrophotometer. The spectra differences betweenthe free-radical substances and their normal tyrosine or tryptophancontaining derivatives were characterized by the appearance of a broadabsorption band with a shoulder at 490 millimicrons in the visibleregion of the spectrum and a shift of the 278 millimicron band in theultnaviolet region to a double peak at 275 and 280 millimicrons withmarked increases in absorbance.

Free-radical measurements were made with a Varian electron spinresonance spectrometer with the amplified signal being passed through a60 cycle notch filter to a computer that averaged transients. ESRmeasurements of the free-radical substances yielded spectrum resultswhich demonstrated a threefold increase in free-radical concentrationwhen the free-radical substances were preliminarily illuminated withvisible light from 1-5 minutes. Various fractions of serum proteins andhormonal peptides yielded free-radical substances with varying spectralintensities depending on the tyrosine and tryptophan content of themolecule. Larger molecules like 7 globulins, myosin and somepreparations of growth hormones tended to crosslin-k and form coloredinsoluble polymers.

Smaller molecules like S-OH tryptophan rapidly turned from a red to adeep brown solution which then stabilized in a soluble free-radicalstate. When stored in solution at 5 C., this S-HTP free-radical slowlyformed an insoluble melanin-like polymer.

Experiments were also performed on 3.5 to 4.0 kgs. adult male albinorabbits with chronically implanted cranial electrodes. The electricalactivity was bipolarly recorded from the anterior part of the parietalcortex. The unfiltered amplitudes of an electroencephalograph weremeasured continuously with an electronic integrator. The values used inthe computations were the cumulative scores of amplitudes obtained onsuccessive periods of one minute each throughout the experimental runs.These.

runs started with a ten minute period during which the animals were keptaroused by varied sensory stimulations. Thereafter, a small dose of Napentobarbital (3 mg./kg. i.v.) was administered in order to induce amild, unspecific, sustained state of sedation. All stimuli were stopped.Five minutes after inception of sedation, the test compound was injectedand the animals were left undisturbed for 30 minutes.

All drug administrations were performed from a distance via a catheterinserted in the marginal ear vein. The animals were carefully trained tothe experimental procedures which took place in a sound attenuatedenclosure. Except for the venous catheter [and the wire connecting theelectrodes to the recording device, the rabbits were not restrained.Drug and control sessions were spaced one week apart.

Variations in the EEG amplitudes were measured in three experimentsperformed on the same animal with normal protein or peptide, with thefree-radical protein or peptide and with the free-radical protein orpeptide illuminated with visible light prior to administration for fiveminutes. The state of sedation followed pentobarbital administration wasmanifested in the EEG by the presence of spindles of high voltage, lowfrequency waves which persisted for an average of 20 minutes after theinjection of physiological saline or normal crystalline serum protein orpeptide. Following administration of the free-radical compounds, a shiftfrom high amplitude, low frequency waves to low amplitude high frequencywaves occurred demonstrating arousal properties. Complete arousalbrought back the wave amplitudes and frequencies to the levelsprevailing prior to sedation with pentobarbital. During the high EEGamplitude recordings, the animals were sitting or lying quietly whileduring the low amplitude recordings, the animals were moving about,searching, licking, etc. When the free-radical substances wereilluminated by light prior to administration, the intensification offree-radical concentration observed by the ESR measurements wasaccompanied by increased arousal and excitatory activities in theanimals.

It has therefore been found that the conversion of naturally occurringmolecules containing tyrosine or tryptophan to a free-radical statespecifically provides arousal and brain excitatory effects in mammalsand apparently generally offers a new approach to the molecularmodification of brain function and a better understanding of thebiochemistry of energy transfer mechanisms in living systems.

Obviously, many modifications and variations of the present inventionare possible in the light of the above teachings. It is therefore to beunderstood that within the scope of the appended claim, the inventionmay be practiced otherwise than as specifically described.

What is claimed is:

1. A method of preparing a stable free-radical product which comprisesthe steps of dissolving three grams of 3X recrystallized bovine serumalbumin in ml. of saline-phosphate buffer (.15 M NaCl, .01 M P0 pH 8.1)to form an albumin solution;

dissolving ."8 gram of fremy salt in 45 ml. of .1 molar Na HPO dilutingsaid fremy salt and .1 molar Na HPO with 100 ml. of water to formnitrosyl disulfonate;

mixing said albumin solution with an equal volume of said nitrosyldisulfonate to form a first product;

standing said first product at 5 C. until said product becomes a colorof deep burgundy red;

precipitating said first product with 3.6 M ammonium sulfate to separatethe protein from the reaction products;

removing the salt from said protein by means of dialysis andchromatography to form said stable free-radical product;

freezing said stable free-radical product at 20 C.

6 References Cited 5 Earland et al.

Chem. Abstracts, vol. 72, 1970 (eifective date October 1969), '64510k,Polis et al.

Polymer. 7(11), pp. 549-556, 1966 Earland et a1.

10 HOWARD E. SCHAIN, Primary Ekaininer US. Cl. X.R.

260-112 B, 112 R, 112 T, 112.7, 999

